BIN2 phosphorylation of GRF5 suppresses low nitrate root foraging by inhibiting transcriptional activity and UBP12/13-mediated deubiquitination

Dataset OverviewThis dataset comprises the raw data supporting the findings in the manuscript entitled "BIN2 suppresses root foraging response under low nitrate by phosphorylating GRF5 to disrupt its interaction with UBP12/13". The data are categorized into four types, which collectively substantiate the key conclusions regarding the BIN2-GRF5-UBP12/13 module in regulating root foraging under low nitrate conditions in Arabidopsis.1. Raw, Uncropped Western Blot ImagesGeneration Process: Data were generated from immunoblotting analyses of protein samples extracted from Arabidopsis thaliana. Proteins were separated by SDS-PAGE or Phos-tag SDS-PAGE, transferred to membranes, incubated with specific antibodies (e.g., anti-GFP, anti-Myc, anti-HA, anti-Ubiquitin), and visualized using enhanced chemiluminescence (ECL).Processing Methods: Images were captured using a gel imaging system and saved as raw, unedited .tif or .jpg files. These images contain all protein bands from the entire membrane, including molecular weight markers. Each raw image corresponds to one immunoblot presented in the main or supplementary figures.Equipment and Tools: Protein electrophoresis and gel imaging were performed using the Bio-Rad electrophoresis and imaging system (Bio-Rad, USA); transfer apparatus.Content Description: Image files are named according to the experiment. They include:Co-IP and pull-down assay results demonstrating BIN2-GRF5 and UBP12/13-GRF5 interactions.Phosphorylation (Phos-tag gel), ubiquitination, and protein stability assays of GRF5 under different genetic backgrounds and nitrate conditions.Actin or total protein loading controls are included for all immunoblots. 2 Representative Microscopy ImagesGeneration Process: Images were captured using confocal, stereomicroscopes, or upright microscopes from Arabidopsis thaliana or Nicotiana benthamiana samples.Processing Methods: Images were captured using microscope-specific software and saved in raw formats such as tif. Fluorescence intensity and staining patterns were not artificially adjusted.Equipment and Tools: Confocal microscope (Zeiss LSM880, Germany), stereomicroscope, upright microscope, photomicrography system.Content Description: Image files are categorized by the experiment type:LUC images: From split-luciferase complementation assays showing interactions (GRF5-BIN2, GRF5-UBP12/13) in N. benthamiana leaves. Luminescence is depicted in red/blue.BiFC images: From bimolecular fluorescence complementation assays showing protein interactions (YFP signal, green) in the nuclei of N. benthamiana epidermal cells (DAPI stained, blue).Root meristem images: Differential interference contrast (DIC) or fluorescent images of Arabidopsis root tips showing cortical cell length in the lateral root elongation zone under normal and low nitrate conditions. GUS staining images: From pBIN2::GUS transgenic plants showing BIN2 expression patterns in root tips (blue staining).Salk primer identification gels: Agarose gel electrophoresis images used to genotype T-DNA insertion mutants (e.g., grf5-1).Root GFP fluorescence images: From transgenic plants expressing GRF5-GFP, showing the subcellular localization of GRF5 in root cells (green).3 Representative Arabidopsis Phenotype ImagesGeneration Process: Seedlings of various genotypes were grown vertically on MGRL solid medium under Normal Nitrate (NN) or Low Nitrate (LN) conditions. After four days of treatment, whole plates were photographed using a digital camera to capture digital images. A fixed stand was used to maintain a consistent distance and angle between the camera and the Petri dish, and standardized lighting was employed to ensure uniform illumination.Processing Methods: Images were captured using a digital camera and saved as raw, uncropped .tif or .jpg files. These images are representative of a single plate from at least three independent experiments.Equipment and Tools: Digital camera (Canon EOS), fixed camera stand, standardized lighting.Content Description: Image files are named by the experiment. They display the whole-root morphology of key genotypes (e.g., Col-0, bin2-1, bin2-3bil1bil2, grf5-1, GRF5OX, UBP12OX) under NN and LN conditions, clearly showing differences in primary and lateral root elongation. 4. Raw Statistical Data (Spreadsheets)Generation Process: Data were obtained from quantitative measurements of the root phenotypes described above, nitrate concentration assays, ¹⁵NO₃⁻ uptake rate assays, and gene expression analyses (qRT-PCR). All raw measurement values were recorded in spreadsheets.Processing Methods: Data were organized using Excel or similar software.Content Description: The file is in .xlsx format with multiple sheets, each corresponding to a statistical panel in the manuscript.Number of Records: Each row represents an independent biological replicate.Column Headers and Meanings:Genotype: Arabidopsis thaliana genotype.Nitrate: Nitrate treatment condition (NN, LN).Primary_Root_Length: Primary root length, measured in centimeters (cm).Avg_LR_Length: Average lateral root length, measured in centimeters (cm).LR_Number: Number of lateral roots.Cortical_Cell_Length: Length of cortical cells, measured in micrometers (μm).Nitrate_Uptake_Rate: ¹⁵NO₃⁻ uptake rate, expressed as μmol ¹⁵N·g⁻¹ DW·h⁻¹.Nitrate_Concentration: Nitrate concentration, expressed as mg/g FW.Relative_Expression: Relative gene expression level determined by qRT-PCR, calculated using the 2^−ΔΔCt^ method with PP2A as an internal control.